An inhibition of p62/SQSTM1 caused autophagic cell death of several human carcinoma cells.

Nihira K, Miki Y, Ono K, Suzuki T, Sasano H - Cancer Sci. (2014)

Bottom Line:
Therefore, in this study, we examined the effects of p62 inhibition on the regulation of autophagy and cell survival in p62-positive carcinoma cells. p62-silencing dramatically suppressed cell proliferation and induced autophagy in p62 expressing PC9 and A549 cells.Electron microscopical analysis revealed the formation of autophagosomes with multilayer membranes caused by p62-silencing. p62 silencing-mediated reduced cell viability was restored by both genomic and pharmacological inhibition of autophagy but not that of apoptosis.These findings were also detected in several types of carcinoma cell lines including adenocarcinomas and squamous cell carcinomas.

fig02: Effects of p62-silencing on autophagic activity. (a) PC9 and A549 cells were transfected with indicated siRNAs. After 3 days, protein homogenates were isolated and immunoblotting was used to measure p62, p-Akt, Akt, p-mTOR, mTOR, LC3B and β-actin. (b) PC9 and A549 cells were treated with ammonium chloride and leupeptin (N/L) or chloroquine (CQ.) and transfected with indicated siRNAs for 3 days. Protein homogenates were isolated and immunoblotting was used to measure p62, LC3B and β-actin. (c) Double immunofluorescent analysis of p62 and LC3B after indicated siRNAs transfection in PC9 cells. At least 100 cells were counted, and frequency of cells with LC3B dots was indicated. Scale bars, 10 μm.

Mentions:
The conversion of LC3B-I into LC3B-II was determined by immunoblotting analysis in order to evaluate the changes of autophagic activities in the cells being treated with two different sip62s. In PC9 and A549 cells, both sip62s inhibited an activation of mTOR (mammalian target of rapamycin; prominent autophagy suppressor) but an activation of Akt, located at the upstream of mTOR signaling pathway, was not changed. In addition, both of these sip62s notably induced the switch of LC3B-I to LC3B-II, indicating that autophagy was activated by both sip62s (Fig.2a). Significant increment of LC3B-II in p62-silenced cells was also detected when treated with lysosomal or autophagic inhibitors (ammonium chloride and Leupeptin or chloroquine, respectively) which indicated that LC3B-II was not simply accumulated but rather induced or synthesized at least partly (Fig.2b). We further confirmed this change using double immunofluorescence evaluations. p62 expression was not detected in p62-silenced cells, and LC3B dots were more frequently detected in p62-silenced cells (Fig.2c). These results all demonstrated that p62 inhibition certainly activated autophagy in PC9 and A549 cells.

fig02: Effects of p62-silencing on autophagic activity. (a) PC9 and A549 cells were transfected with indicated siRNAs. After 3 days, protein homogenates were isolated and immunoblotting was used to measure p62, p-Akt, Akt, p-mTOR, mTOR, LC3B and β-actin. (b) PC9 and A549 cells were treated with ammonium chloride and leupeptin (N/L) or chloroquine (CQ.) and transfected with indicated siRNAs for 3 days. Protein homogenates were isolated and immunoblotting was used to measure p62, LC3B and β-actin. (c) Double immunofluorescent analysis of p62 and LC3B after indicated siRNAs transfection in PC9 cells. At least 100 cells were counted, and frequency of cells with LC3B dots was indicated. Scale bars, 10 μm.

Mentions:
The conversion of LC3B-I into LC3B-II was determined by immunoblotting analysis in order to evaluate the changes of autophagic activities in the cells being treated with two different sip62s. In PC9 and A549 cells, both sip62s inhibited an activation of mTOR (mammalian target of rapamycin; prominent autophagy suppressor) but an activation of Akt, located at the upstream of mTOR signaling pathway, was not changed. In addition, both of these sip62s notably induced the switch of LC3B-I to LC3B-II, indicating that autophagy was activated by both sip62s (Fig.2a). Significant increment of LC3B-II in p62-silenced cells was also detected when treated with lysosomal or autophagic inhibitors (ammonium chloride and Leupeptin or chloroquine, respectively) which indicated that LC3B-II was not simply accumulated but rather induced or synthesized at least partly (Fig.2b). We further confirmed this change using double immunofluorescence evaluations. p62 expression was not detected in p62-silenced cells, and LC3B dots were more frequently detected in p62-silenced cells (Fig.2c). These results all demonstrated that p62 inhibition certainly activated autophagy in PC9 and A549 cells.

Bottom Line:
Therefore, in this study, we examined the effects of p62 inhibition on the regulation of autophagy and cell survival in p62-positive carcinoma cells. p62-silencing dramatically suppressed cell proliferation and induced autophagy in p62 expressing PC9 and A549 cells.Electron microscopical analysis revealed the formation of autophagosomes with multilayer membranes caused by p62-silencing. p62 silencing-mediated reduced cell viability was restored by both genomic and pharmacological inhibition of autophagy but not that of apoptosis.These findings were also detected in several types of carcinoma cell lines including adenocarcinomas and squamous cell carcinomas.